The search for the cause and effect of genetic changes associated with neoplastic cells occupies a central place in cancer research. A year and a half ago, we began to address the possible causes of a human laryngeal cancer. We have now reported the association of an abnormal RAF oncogene with a radiation resistant human laryngeal carcinoma cell line, SQ-20B. Our studies have been complemented by the independent observations made in another laboratory that noncancerous, radiation resistant skin fibroblasts of patients with a familial history of radiation resistant malignancies reveal an abnormal RAF oncogene. However, these two reports have not yet proven the precise nature of this abnormality, nor its relevance to the resistance of target cells to radiation toxicity. Our major gaols during the tenure of this grant period will be to study the modulation of RAF gene expression and the cellular responses to radiation-damage as a consequence of delivering an optimal expression of sense or anti-sense RAF to the appropriate mammalian cells (AIM I), to dissect and characterize the RAF oncogene structure and function in SQ-20B cells (AIM II), and to search for genetic abnormalities in three additional closely related human cancers (AIM III). We will use a clearly defined, feasible, and integrated approach to evaluate the genetic basis of malfunctioning of the RAF in SQ- 20B cells. These studies are greatly facilitated by the availability of cloned and defined unique expression vectors containing human RAF(sense/anti-sense) sequence, c-RAF-1 DNA probes, riboprobe vector probes suitable for RAF (anti-sense+) analysis, and antibody to RAF protein from Dr. George Mark (NIH). We will maintain our ongoing collaborative efforts with Drs. Anatoly Dritschilo, George Mark, and Michael Gottesman for their specific expertise in the areas of radiobiology, molecular biology of RAF, and molecular biology of multidrug resistance, respectively. Finally, the current investigations should lead us to begin to elucidate the structure and function of RAF proto- oncogene domains pivotal to the maintenance of normal cell proliferation and behavior.